This proposal addresses a fundamental aspect of post-transcriptional control in eukaryotes: the molecular mechanisms that regulate mRNA decay. At least three different types of events trigger the initiation of mRNA decay, including poly(A) shortening, endonucleolytic cleavage, and the arrest of translation at a premature nonsense codon. Using the yeast Saccharomyces cerevisiae as a model system, work in my laboratory has shown that the latter mechanism, nonsense-mediated mRNA decay, requires a sequence downstream of the nonsense codon (the "downstream element"), is inhibited by ribosome traversal of a second element (the "stabilizer"), and is dependent on the activity of several factors, including Upf1p, Nmd2p, Upf3p, Nmd3p, and Dbp2p. We have also shown that the downstream element is a site of translational reinitiation, an event that requires the presence of the nonsense codon and the UPF1, NMD2, and UPF3 gene products. In this proposal, I seek to test hypotheses that reinitiation triggers mRNA decay, that the stabilizer governs the association of specific decay factors with the ribosome, and that there may be a nuclear component to this predominantly cytoplasmic pathway. We will: 1) further characterize the interactions of Upf1p, Nmd2p, and Upf3p with each other and with other proteins and test whether such interactions are required for mRNA decay; 2) determine whether the factor and mRNA sequence requirements for decay coincide with those required for reinitiation; 3) define the mRNA sequences essential for stabilizer function and determine their influence on the presence or absence of specific factors on individual polysomes and mRNPs; and 4) assess a possible association of Nmd2p and other factors with the nucleus and with newly synthesized mRNA.